The dopamine transporter (DAT) mediates the inactivation of released dopamine (DA) through its reuptake. The psychostimulant amphetamine (AMPH), by acting as a DAT substrate, promotes reversal of DA transport (DA efflux) and increases extracellular DA levels, an event of importance for the stimulant properties of AMPH. During the previous project period, we demonstrated that the SNARE protein syntaxin1 (Stx1) that is enriched in membrane rafts, associates with the DAT N-terminus, and that this association regulates AMPH-induced DA efflux. We have also shown that the membrane raft-associated protein Flotillin1 (Flot1) is necessary for the localization of DAT to membrane rafts. Flot1 knockdown, which displaces DAT from membrane rafts, blunts AMPH-induced DA efflux. This proposal aims to uncover the mechanisms by which DAT localization in membrane rafts regulates DAT function, DA efflux, and importantly, AMPH-induced behaviors. Phosphatidylinositol-4,5-bisphosphate (PIP2) is a key phospholipid that is mainly concentrated in the inner leaflet of the plasma membrane and is enriched in lipid raft domains. In addition to other functions, PIP2 acts as an essential cofactor to mediate protein recruitment and localization. This includes Stx1, which is phosphorylated at Ser14 by casein kinase 2 (CK2). We have shown that Stx1 binds the DAT N-terminus, and that AMPH promotes this interaction. Preliminary results suggest that PIP2 coordinates Stx1 phosphorylation, DAT/Stx1 associations, as well as DA efflux. They also suggest that AMPH stimulates CK2-mediated Stx1 phosphorylation, a molecular event that we hypothesized to be required for DAT/Stx1 interactions as well as DA efflux. Consistent with this hypothesis is evidence that suggests that inhibition of CK2 reduces Stx1 phosphorylation and DA efflux. We have established DAT-mediated behavioral assays (grooming and locomotion) in Drosophila melanogaster to determine whether and how AMPH-induced behaviors are dictated by the molecular elements and mechanisms we hypothesize support DA efflux. Our preliminary data suggest that in Drosophila, deletion of DAT or knockdown of CK2 in DA neurons inhibits AMPH-induced behaviors. The long-term goals of this research are to understand how AMPH-induced DA efflux and associated behaviors are dictated by DAT interactions with Stx1 and PIP2, and/or by post-translational modifications of DAT and DAT-associated proteins. These hypotheses will be tested (for the first time) in vitro in synaptic boutons, and in vivo in Drosophila melanogaster.